HRMAS13C NMR and genome-scale metabolic modeling identify threonine as a preferred dual redox substrate forClostridioides difficile

Abstract

AbstractStickland-fermentingClostridiapreferentially ferment amino acids to generate energy and anabolic substrates for growth. In gut ecosystems, these species prefer dual redox substrates, particularly mucin-abundant leucine. Here, we establish how theronine, a more prevalent, mucin-abundant substrate, supports dual redox metabolism in the pathogenClostridioides difficile. Real-time, High-Resolution Magic Angle Spinning NMR spectroscopy, with dynamic flux balance analyses, inferred dynamic recruitment of four distinct threonine fermentation pathways, including ones with intermediate accrual that supported changing cellular needs for energy, redox metabolism, nitrogen cycling, and growth. Model predictions with13C isotopomer analyses of [U-13C]threonine metabolites inferred threonine’s reduction to butyrate through the reductive leucine pathway, a finding confirmed by deletion of thehadA2-hydroxyisocaproate CoA transferase.In vivometabolomic and metatranscriptomic analyses illustrate how threonine metabolism inC. difficileand the protective commensalParaclostridium bifermentansimpacts pathogen colonization and growth, expanding the range of dual-redox substrates that modulate host risks for disease.